Vaccination strategies have failed to impact outcomes of cancer patients significantly! Why? We hypothesize that a primary reason for the failure of tumor vaccine strategies is because the magnitude of the antitumor immune response is insufficient to mediate tumor regression. Our own work, as well as that of others, has identified an approach that exploits the increased sensitivity of lymphocytes to respond to antigenic stimulae when they are placed under conditions of homeostasis-driven proliferation. We modeled this by vaccinating lymphopenic mice with a GM-CSF gene-modified melanoma cell line (D5-G6) following reconstitution with naive spleen cells. Subsequent examination of tumor vaccine-draining lymph node (TVDLN) T cells from reconstituted-lymphopenic mice (RLM) revealed a substantial increase in the frequency of activated T cells. Following in vitro activation these T cells contained a significantly elevated frequency of tumor- specific CD4 and CD8 T cells with augmented function in vitro and therapeutic efficacy in vivo. When reconstitution was performed using spleen cells from systemic B16BL6-D5 (D5) tumor-bearing mice (TBM), anti-tumor function was lost. However, depletion of CD25+ Treg cells from the spleen cells of TBM used for reconstitution led to recovery of tumor-specific function and therapeutic efficacy. This proposal builds on these findings, our extensive preclinical and clinical data to propose two clinical trials. The first, Aim 1, is a randomized Phase II trial that will directly translate our preclinical observations and determine whether lymphopenic patients vaccinated with autologous tumor and GM-CSF, and reconstituted with either autologous total PBMC or CD25-depleted PBMC will generate a higher frequency of tumor-specific IFN-gamma secreting T cells. This trial will allow us to "pick the winner". The second trial, Aim 4, will use the winning strategy to induce tumor-specific T cells that will be harvested, activated and adoptively transferred back to the patient. Aim 2 will characterize changes in the effector, effector/memory and central memory phenotype of tumor-specific T cells of patients treated on clinical trials proposed in aims 1 and 4. Aim 3 will characterize the regulatory T cell population in the peripheral blood prior to and following induction of lymphopenia and reconstitution.